Our laboratory is elucidating the nature of the immune response to tumors and establishing principles which may enhance our ability to immunize patients against known tumor antigens and induce tumor regression with cytokine or cell-transfer therapies. We have developed methodologies for identifying tumor-specific CTL from patients with renal cell cancer and successfully used these techniques to identify unmutated FGF-5 as an HLA-A3 restricted antigen on RCC, recognized by T-cells. This target protein has significant limitations on its clinical utility, such that the discovery of alternative tumor antigens is needed. Continuing these efforts looking at other antigens expressed on RCC, we have found tumor-reactive T-cells in patients with RCC which recognize their own tumors as well as allogeneic RCCs. Many of these cells are CD4 cells, recognizing antigens presented by HLA Class II molecules. The role and efficacy of tumor-reactive CD4+ cells in tumor rejection is still unclear. A more extensive catalog of such antigens will not only clarify our basic understanding of the role of CD4+ cells, but allow the recruitment of this other arm of the cellular immune response against cancer in novel immunotherapies. Renal cancer serves as a particularly useful model, because, unlike melanoma, the preponderance of tumor reactivity seems to reside in the CD4 compartment, yet this cancer is also quite susceptible to immunotherapies which can also treat melanoma (which is dominated by CD8 reactivity). We have developed a system of in vitro lymphocyte stimulation with autologous dendritic cells and autologous tumor which consistently generates tumor-reactive T-cells. We are adapting methods for cloning the antigens recognized to further our understanding of tumor recognition by CD4 cells. The initial phase of this effort is to devise improved methods for identifying and purifying T-cells with tumor reactivity from bulk populations of immune cells generated in vitro or procured from tumor infiltrating lymphocytes. This uses phenotypical markers as well as novel reporter genes indicating immunoreactivity. Manipulation of the conditions of T-cell sensitization by modulating costimulatory molecules will also be employed. The second portion of the project is to select the most avid clones with tumor reactivity generated in phase I and clone their T-cell receptors. These then are introduced into retroviral or lentiviral expression vectors for use in gene therapy approaches to generate T-cells for clinical adminstration.